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A Review on Sampling Techniques and Analytical Methods for Microbiota of

Cultural Properties and Historical Architecture

Article in Applied Sciences · November 2020

DOI: 10.3390/app10228099

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 applied
sciences
Review
A Review on Sampling Techniques and Analytical
Methods for Microbiota of Cultural Properties and
Historical Architecture
Xinghua Ding 1 , Wensheng Lan 2 and Ji-Dong Gu 1, *,†
1 Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences,
The University of Hong Kong, Pokfulam Road, Hong Kong, China; [email protected]
2 Shenzhen R&D Key Laboratory of Alien Pest Detection Technology, The Shenzhen Academy of Inspection
and Quarantine, Technology Center for Animal and Plant Inspection and Quarantine, Shenzhen Customs,
1011 Fuqiang Road, Shenzhen 518010, China; [email protected]
* Correspondence: [email protected]
† Current address: Environmental Engineering, Guangdong Technion Israel Institute of Technology,
241 Daxue Road, Shantou 515063, Guangdong, China.




Received: 28 September 2020; Accepted: 20 October 2020; Published: 16 November 2020


Abstract: World cultural heritage suffers from deterioration caused by both natural and anthropogenic
processes, among which microorganisms are significantly involved. Among the key issues of this
topic, sampling techniques and analytical methods for revealing the microbiome are fundamental to
obtaining useful results for understanding the key players and processes involved, and also for effective
protection and management of the cultural heritage for humanity. A non-invasive and non-destructive
sampling method is required for sampling of cultural properties prior to further analysis of the
microbiome. One example is illustrated in this article. For many years, culture-dependent methods
had been used before the invention of polymerase-chain reaction (PCR) methods and, more recently,
specifically high-throughput next generation sequencing (NGS). NGS reveals the whole microbial
community composition and the active microorganisms from genomic DNA and RNA, respectively.
The recovered environmental DNA and RNA from samples provide the information on microbial
community and composition, and the active members and biochemical processes of the microbial
attributes. It should be emphasized that the metabolically-active members of functional microflora in
the biofilm or microbiome on cultural heritage must be determined and identified from the RNA-based
analysis to gain a substantially important insight of the active biodeterioration processes and also the
effectiveness of the conservation strategies. The importance of the culture-independent technique,
based on NGS, is that it can be used in combination with the conventional culturing methods to
guide the isolation and enrichment of new microorganisms to gain further biochemical insights to
advance the role of the specific microbial groups for biodeterioration of cultural heritage. At the same
time, effective restoration and maintenance strategies can be formulated for the protection of world
cultural heritage.

Keywords: cultural heritage; microbiome; biochemical processes; nitrogen cycle; sulfur cycle; acidic
attack; stone and rock

1. Introduction
Protection of cultural heritage, a social ‘resource’, has been recognized by many countries as both
an economic asset and also an important factor for promotion of social integration [1]. At the same
time, world cultural heritage and archaeological sites are the bases for the global tourism industry and
economy [2–4]. World cultural heritage is broadly defined by including physical or “tangible” assets,

Appl. Sci. 2020, 10, 8099; doi:10.3390/app10228099 www.mdpi.com/journal/applsci

Appl. Sci. 2020, 10, 8099 2 of 15

including archaeological sites, architecture, monuments, buildings, sculptures etc. and intangible
attributes, such as performing arts, oral traditions, rituals etc. Cultural heritage can be physically
movable, e.g., books, documents, movable artworks, machines, clothing, and other artifacts worthy of
preservation for the future. Meanwhile, the physical non-movable heritage of buildings, architectural
ornaments, other historic sites, and monuments is the legacy of history and humanity, allowing selective
value to pass from generation to generation. The recognition of the necessity of the historical value of
the civilization and of the objects that possess their value and significance promotes the necessity for
preservation of culture and heritage for conservation and protection through scientific understanding
and better management [5–16].
Conservation science studies the processes that lead to physical changes in historical objects or
artifacts that affect their longevity and integrity as a result of various processes or mechanisms of
deterioration. These deteriorative mechanisms inform the scientists or conservators about the potential
risk factors and strategies to mitigate adverse environmental impacts through management policy
to effectively assist long-term conservation [17–22]. Conservation science has gradually evolved into
an interdisciplinary research subject by embracing physics, chemistry and biology, microbiology,
engineering, materials science, and engineering as well as archaeology, anthropology, and art
history [23–28]. In the current conservation research, significant efforts are being made by non-biological
analyses, mostly chemistry and material characterization, while an emerging direction has been in
analyzing microorganisms on these objects for a more comprehensive knowledge and preservation
practice [29,30]. Fungi, bacteria, archaea, and lichens have been observed on a variety of historic artifacts
such as mural paintings and bas-reliefs in caves, churches and catacombs, stone monuments and
architectural surfaces in outdoor environments, and historical/archaeological relics and sites [31–39].
The biodeterioration caused by microorganisms not only influences the aesthetical appearance
of the cultural heritage, but also sometimes results in destruction of the structural integrity of
the valuable cultural heritage via (bio)chemical processes such as acidification, salt crystallization,
calcium carbonate precipitation, water retention, accumulation of pollutants through biofilm formation,
and progression [30,38,40–42]. As the world cultural heritage is both unique and irreplaceable, is hardly
possible to estimate the economic value of the damage caused by microbial degradation.
Microbiological research on this subject is becoming recognized as an indispensable part of the
overall conservation science and the application of current scientific analytical techniques enables
microbiologists to determine the microbial community and composition as well as biodeterioration
potential of the colonizing microorganisms on the cultural heritage under a given environmental
condition. Such knowledge informs how deterioration is likely to occur on cultural heritage and reveals
important clues for conservators to monitor or mitigate the adverse effects and to prevent the potential
risk from deterioration of historical artworks and objects caused by microorganisms [43,44]. This article
offers a synthesis of the important issues of microbiological research for cultural heritage conservation
by focusing specifically on sampling and new analytical methods. Furthermore, an outlook is given on
the future research needs and developments in this highly important and an emerging research field.

2. Sampling Techniques
With respect to cultural heritage, conservation science focuses mainly on protection and
maintenance of physical or “tangible” cultural heritage such as artworks, architecture, monuments,
and museum collections. The code of ethics in the conservation of cultural heritage outlines that
the conservation–restoration of any given physical or “tangible” heritage is to only do the minimum
required for preservation [45]. Thus, a detailed diagnostic analysis of the current status of a cultural
heritage object by a non-invasive/non-destructive technique is a prerequisite to avoiding unnecessary
or potential damage and to keeping the amount of handling to a minimum. Before the advent of the
specialized instruments used for conservation studies, visual inspections were the only non-invasive
inspection method available in the field of heritage conservation to examine for visual detectable signs
of damage, decay, or deterioration. Because of the methodological limitations of visual inspection,
Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 16

non-invasive inspection method available in the field of heritage conservation to examine for visual
detectable signs of damage, decay, or deterioration. Because of the methodological limitations of
Appl. Sci. 2020, 10, 8099 3 of 15
visual inspection, the earliest conservation studies could not yield the detailed quantitative
information on deterioration processes and mechanisms, and always led to inconclusive results
[46,47]. Microorganisms
the earliest were not
conservation studies previously
could not yield considered
the detailed in this field. Currently,
quantitative information a number of generic
on deterioration
or specialized research techniques have begun to be applied to heritage
processes and mechanisms, and always led to inconclusive results [46,47]. Microorganisms were not conservation studies to
provide
previously theconsidered
powerful meansin thisto analyze
field. the current
Currently, state of
a number of generic
an object orfor preservation
specialized or to techniques
research detect any
products of deterioration and decay that ought to be removed or
have begun to be applied to heritage conservation studies to provide the powerful means to analyze cleaned. On the collection of
samples
the current involved
state ofinanheritage
object for conservation
preservation studies, the dogma
or to detect which outlines
any products the best practice
of deterioration and decay for
conservators and scientists alike for scientific analysis is “non-invasive
that ought to be removed or cleaned. On the collection of samples involved in heritage conservation and non-destructive method
for bio-molecular
studies, the dogma and whichchemical
outlines theanalysis that requires
best practice very small
for conservators andquantities
scientists alikeof sample from
for scientific
inconspicuous locations on
analysis is “non-invasive artworks
and and objects”
non-destructive [48]. for
method As bio-molecular
the requirement and forchemical
sampling techniques
analysis that
has suggested, any analytical techniques to be used must be highly
requires very small quantities of sample from inconspicuous locations on artworks and objects” sensitive, requiring minimum [48].
amounts
As of sample for
the requirement or none
samplingat all. Because of
techniques hasthis, conservation
suggested, microbiologists
any analytical techniques need to to
bemaintain
used must a
close communication
be highly with conservators,
sensitive, requiring minimum amounts curators, and heritage
of sample or none at authorities
all. Because and workconservation
of this, closely on
research
microbiologists need to maintain a close communication with conservators, curators, andproposed
projects to determine the most appropriate sampling schemes and the heritage
measurements
authorities and prior work to carryon
closely outresearch
the sampling.
projects to determine the most appropriate sampling schemes
A significant
and the proportion of the
proposed measurements priorcultural
to carry heritage
out therelics and physical objects that suffer varying
sampling.
levelsAof biodeterioration caused by microorganisms
significant proportion of the cultural heritage relics and have been rescued
physical by salvage
objects archaeology
that suffer varying
effort. Under non-ideal preservation and storage conditions, artifacts
levels of biodeterioration caused by microorganisms have been rescued by salvage archaeology that have always been in
effort.
storage such as archives, books, documents and paintings, and
Under non-ideal preservation and storage conditions, artifacts that have always been in storagetheir constituent materials, both
organic
such as and inorganic,
archives, books,can provide aand
documents suitable surface
paintings, andor their
substratum for colonization
constituent materials, both by different
organic
types of microorganisms to form biofilm [30,38,49,50]. Microorganisms
and inorganic, can provide a suitable surface or substratum for colonization by different types grow on the surface of
artifacts mainly in clusters or aggregates called microbial biofilms,
of microorganisms to form biofilm [30,38,49,50]. Microorganisms grow on the surface of artifacts that have the potential to cause
adverse
mainly in reactions
clusterschemically
or aggregates or biochemically
called microbial with culturalthat
biofilms, heritage
have theobjects [30,31,36].
potential to causeA microbial
adverse
biofilm is a collection of microbial cells and extracellular polymer
reactions chemically or biochemically with cultural heritage objects [30,31,36]. A microbial biofilm substances (EPSs) together is
adhering
a collection onto a solid substratum
of microbial cells and surface (Figure
extracellular 1). Theysubstances
polymer are ubiquitous
(EPSs) and play anadhering
together important rolea
onto
in
solidecosystems.
substratumNumeroussurface (Figure studies haveareconfirmed
1). They ubiquitousthat biofilms
and play are not role
an important simple microbial
in ecosystems.
assemblages, but have well-defined spatial distribution characteristics
Numerous studies have confirmed that biofilms are not simple microbial assemblages, but have and microbial community
composition spatial
well-defined and structures
distributiontocharacteristics
assist in their and ecological functions composition
microbial community [33,51]. Spatial microbial
and structures
distribution
to assist in theirhasecological
been reported in some
functions [33,51].environmental
Spatial microbial biofilms, especially
distribution has inbeenaquatic
reported ecosystems
in some
[52–54]. Microorganisms communicate via chemical cues prior to
environmental biofilms, especially in aquatic ecosystems [52–54]. Microorganisms communicate and during the biofilm formation.via
A stratified bacterial structural organization exhibited in biofilms has been
chemical cues prior to and during the biofilm formation. A stratified bacterial structural organization reported on the limestone
of historical
exhibited monuments
in biofilms at a Mayan
has been reported siteon[55],
the and on sandstone
limestone of Angkor
of historical monumentstemples at awith
Mayan bothsite
spatial
[55],
and temporal dynamics [33,56]. These adverse (bio)chemical reactions
and on sandstone of Angkor temples with both spatial and temporal dynamics [33,56]. These adverse range from simply stains to
acidification,
(bio)chemical discoloration,
reactions rangeand fromutilization
simply stainsof thetomaterials
acidification,as nutrients and weaken
discoloration, the structure
and utilization of the
causing further disintegration and delamination.
materials as nutrients and weaken the structure causing further disintegration and delamination.

Figure 1. A SEM micrograph showing a matured biofilm consisting of both grown cells and extracellular
polymeric materials on surface of a rock under moist conditions (scale bar, 2 µm).
 Appl. Sci. 2020, 10, x FOR PEER REVIEW 4 of 16

Figure 1. A SEM micrograph showing a matured biofilm consisting of both grown cells and
extracellular
Appl. Sci. polymeric materials on surface of a rock under moist conditions (scale bar, 2 μm).
2020, 10, 8099 4 of 15

Earlier sampling techniques required relatively large amount of materials to be taken from the
object before
Earlier chemicaltechniques
sampling and especially microbiological
required relatively analyses couldof
large amount bematerials
carried out todue to thefrom
be taken lowerthe
sensitivity
object beforeofchemical
the analytical techniques
and especially involved. Because
microbiological of this,
analyses the be
could focus of this
carried outarticle
due tois the
on lower
the
current sampling techniques with a minimum and non-invasive approach
sensitivity of the analytical techniques involved. Because of this, the focus of this article is on the current coupled with
highly-sensitive
sampling techniquesmicrobiological
with a minimum analysis.
andCurrently,
non-invasive a slightly
approach different
coupled version of the same
with highly-sensitive
principle has been
microbiological developed
analysis. as a sampling
Currently, a slightly technique using sterilized
different version of the sameplastic adhesive
principle sheetdeveloped
has been or tape
to remove microbial biofilms and loosely-attached materials including degradation
as a sampling technique using sterilized plastic adhesive sheet or tape to remove microbial biofilms products from
surfaces, regardless of the surface physical morphologies and material types
and loosely-attached materials including degradation products from surfaces, regardless of the surface [57] (Figure 2). This
sampling device was initially developed by the Japan Space Agency for
physical morphologies and material types [57] (Figure 2). This sampling device was initially developedsampling surfaces for
bymicrobiological
the Japan Space assessment on the International Space Station and then it was then adopted in the
Agency for sampling surfaces for microbiological assessment on the International
cultural heritage conservation work at Angkor for more than a couple of decades [31,33,58–62]. This
Space Station and then it was then adopted in the cultural heritage conservation work at Angkor for
sampling technique, with some small variations, has also been used in Europe for microbiological
more than a couple of decades [31,33,58–62]. This sampling technique, with some small variations,
analysis of cultural heritage (Urzi and De Leo, 2001). The device, consisting of an adhesive on a
has also been used in Europe for microbiological analysis of cultural heritage (Urzi and De Leo,
plastic sheet and a supporting paperboard, is made under sterile conditions, and can be
2001). The device, consisting of an adhesive on a plastic sheet and a supporting paperboard, is made
conveniently carried to sampling locations. At the sampling site, after peeling the paperboard from
under sterile conditions, and can be conveniently carried to sampling locations. At the sampling site,
the adhesive and attaching the adhesive surface to the sampling location to obtain the materials from
after
the peeling the paperboard
surface onto adhesive (Figure from the3), adhesive and attaching analyses
further microbiological the adhesivecan be surface
carriedto the
out sampling
on the
location to obtain the materials from the surface onto adhesive (Figure
materials picked up on the adhesive. This sampling technique shows a higher recovery rate for 3), further microbiological
analyses
microbialcanpopulations
be carried out on the
than the materials picked
conventional up onswab
cotton the adhesive.
method [63] This or
sampling
stamp technique
agar [57].shows
By
a applying
higher recovery
the adhesive technique in sampling, samples could be collected as many timesmethod
rate for microbial populations than the conventional cotton swab [63] or
as required
stamp agar [57].
at a selected By applying
location to obtainthe spatial
adhesive technique information
distribution in sampling,and samples could beofcollected
composition as many
the microbial
times as required at a selected location to obtain spatial distribution
community from the sandstone bas-relief wall of Bayon temple of Angkor Thom in Cambodia information and composition
of[31,33].
the microbial community
Specifically, samplesfrom werethe sandstone
analyzed bas-relief
to establish thewall of Bayonbetween
relationship temple the of Angkor
specific Thom
colors in
Cambodia [31,33]. Specifically,
on the sandstone wall and the samples weremicroorganisms
dominant analyzed to establish[33]. the
By relationship
working closely between
withthe specific
Apsara
colors on theand
Authority sandstone wall and
the Ministry theEnvironment,
of the dominant microorganisms [33]. By working
Kingdom of Cambodia, closely with
this sampling Apsara
technique
was systematically
Authority appliedof
and the Ministry in the
the Environment,
conservation and protection
Kingdom research ofthis
of Cambodia, Angkor
samplingmonuments
techniqueoverwas
the past 20 years
systematically or more
applied in thetoconservation
accumulate data on multipleresearch
and protection dimensions of the monuments
of Angkor research objectives
over the
[31,33,51].
past 20 years or more to accumulate data on multiple dimensions of the research objectives [31,33,51].

Figure2.2.The
Figure Theadhesive
adhesive sheet
sheet sampling devicedeveloped
sampling device developedfor fornon-invasive/non-destructive
non-invasive/non-destructive sampling
sampling
from various solid surfaces. The adhesive sheet can collect biofilm (the microbial community
from various solid surfaces. The adhesive sheet can collect biofilm (the microbial community and the and
the decomposed
decomposed materials
materials on on
thethe solid
solid surface)
surface) from
from thethe surface
surface to the
to the deepest
deepest layer
layer by by a series
a series of of
continuous
continuouspeeling-off
peeling-offsampling
sampling of
of the biofilm at
the biofilm atthe
thesame
samesampling
samplingposition
position
toto analyze
analyze thethe stratified
stratified
microbiome
microbiomestructure.
structure.
Appl. Sci. 2020, 10, 8099 5 of 15
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 16

Figure 3. The adhesive sampling device in a slightly different configuration from that shown in Figure 2
Figure 3. The adhesive sampling device in a slightly different configuration from that shown in
used for sampling the surface colonizers on sandstone wall of Angkor temple in Cambodia and then
Figure 2 used for sampling the surface colonizers on sandstone wall of Angkor temple in Cambodia
transferred into a sterile plastic bag ready for preservation and transportation.
and then transferred into a sterile plastic bag ready for preservation and transportation.
The adhesive sampling technique allows analysis of samples for microbial community and
The
composition adhesive
on thesesampling
sheets technique
afterward allows
[31,33].analysis of samples
For example, for microbial community
the characteristics and
of the stratified
composition on these sheets afterward [31,33]. For example, the characteristics
structure of bacteria in the biofilm were analyzed and characterized by using 16S rRNA-based of the stratified
structure of bacteria
PCR-denaturing in the
gradient gelbiofilm were analyzed
electrophoresis (DGGE)and characterized
analyses by usingcommunities
among bacterial 16S rRNA-based after
PCR-denaturing gradient gel electrophoresis (DGGE) analyses among
non-destructive collection of biofilm samples onto adhesive sheets from the sandstone surface bacterial communities after
of
non-destructive collection of biofilm samples onto adhesive sheets from
Bayon temple at Angkor Thom, Cambodia [33]. The study disclosed a rich community of bacteria, the sandstone surface of
Bayon temple
especially at Angkor Thom, Cambodia
cyanobacteria-related bacteria, on[33].
the The
outerstudy
surfacedisclosed
layer ofa the
richbiofilm,
community of bacteria,
but fewer in the
especially cyanobacteria-related bacteria, on the outer surface layer of the biofilm,
lower layer; while Chloroflexi-related bacteria appeared only in the lower layer of the biofilm. A similar but fewer in the
lower layer;ofwhile
distribution Chloroflexi-related
bacterial bacteria
structure has already beenappeared
reported in only in the mats
microbial lowerinlayer
a hot of the biofilm.
spring which was A
similar
very richdistribution of bacterial
in cyanobacteria structure
near the has
surface already
and been reported
Chloroflexi in deeperinlayers
microbial matsConsidering
[64–67]. in a hot springthe
which was very rich in cyanobacteria near the surface and Chloroflexi
differences between the optical absorption spectra of the Chloroflexi and Cyanobacteria together in deeper layers [64–67].
with
Considering the differences
the phylum Chloroflexi, whichbetween the of
are an order optical absorption
heterotrophic spectra
bacteria andofanthe order Chloroflexi
of anoxygenicand
Cyanobacteria together with the phylum Chloroflexi, which are an order of heterotrophic
phototrophs [67–69], these results suggest that the bacteria residing within the biofilm are very specific bacteria
and an order
spatially, of anoxygenic
depending phototrophs
on the availability [67–69],
of light and these
organic results suggest
nutrients and that the bacteria
the oxygen residing
concentration.
within
The the biofilm
biofilm formation are andverydevelopment
specific spatially, depending
on cultural heritage on surfaces
the availability
affect not of only
light the
andaesthetic
organic
nutrients and the oxygen concentration. The biofilm formation and development
appearance of the artistic presentation and clarity but also the integrity of its underling materials on cultural
heritage surfaces affect
by the colonization of not only the aesthetic
microorganisms, and appearance of the artistic
biofilm formation presentation [30,31,40,49,70].
and development and clarity but
also the integrity
Information of its
about the underling
spatial materials
distribution by the colonization
characteristics and microbial of compositional
microorganisms, and biofilm
structures of the
formation and development [30,31,40,49,70]. Information
microbial communities in the biofilm is therefore indispensable for understanding the about the spatial distribution
microbial
characteristics
community involved and microbial
in thecompositional
biofilm formationstructures
and of thecontrolling
also microbial communities in the biofilm
biofilm colonization of theis
therefore indispensable
cultural heritage. for understanding the microbial community involved in the biofilm
formation and also controlling biofilm colonization of the cultural heritage.
3. Microbiome Analysis by Culture-Independent Methods
3. Microbiome Analysis by Culture-Independent Methods
Knowledge of the microorganisms inhabiting cultural heritage and their involvement in the
Knowledge
material of theismicroorganisms
deterioration inhabiting
basic for conservators cultural actions
to formulate heritagetoand their and
preserve involvement in the
protect cultural
material
heritage.deterioration
For decades, is basic for conservators
culture-dependent to formulate
approaches have been actions to preserve
applied to isolateandand protect cultural
identity any
heritage. For decades, culture-dependent approaches have been applied
culturable microorganisms from the microbiome inhabiting cultural heritage [60–62,71,72], but thisto isolate and identity any
culturable
approach has microorganisms
many shortcomings. from theManymicrobiome inhabiting cultural
naturally-occurring heritage [60–62,71,72],
microorganisms but this
that can be cultured
approach has many shortcomings. Many naturally-occurring microorganisms
in laboratory media are selected by the medium composition and also the culturing conditions [73], that can be cultured in
laboratory media are selected by the medium composition and also the
not the dominant members in the niche or in situ conditions, so the subsequent results of the culturing conditions [73], not
the dominant members in the niche or in situ conditions, so the subsequent results of the physiology
and biochemistry of these microorganisms have very limited relevance or value to the whole
Appl. Sci. 2020, 10, 8099 6 of 15

Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 16

physiology and biochemistry of these microorganisms have very limited relevance or value to the whole
community
communitycomposition
composition and the the
and biochemical processed
biochemical involved.
processed In addition,
involved. microorganisms
In addition, cultured
microorganisms
under laboratory
cultured conditionsconditions
under laboratory experience much higher
experience much concentrations of nutrients
higher concentrations than the ambient
of nutrients than the
environmental conditions
ambient environmental on the cultural
conditions on the heritage, and this critical
cultural heritage, and thisfactor hasfactor
critical been has
largely
beenignored
largely
in practice (Figure 4). The microbiome on sandstone is dominated by halophile
ignored in practice (Figure 4). The microbiome on sandstone is dominated by halophile and and microorganisms
are capable of adaptation
microorganisms to the
are capable local conditions
of adaptation to theand also
local enter a physiological
conditions and also enter state, called viablestate,
a physiological but
non-culturable (VNC) state under adverse condition of nutrient limitation and low temperature
called viable but non-culturable (VNC) state under adverse condition of nutrient limitation and low [74,75].
Under natural[74,75].
temperature condition,
Under a complex microbial community
natural condition, is the normal
a complex microbial state rather
community is thethan the pure
normal state
culture of a single
rather than species
the pure in artificial
culture medium.
of a single Theinhalophilic
species artificial condition
medium. on The sandstone
halophilic selects relevant
condition on
microorganisms and similarly antibiotic-resistant populations can be developed
sandstone selects relevant microorganisms and similarly antibiotic-resistant populations can beafter such chemicals
have been applied
developed for chemicals
after such a while to have
alter the
beenindigenous
applied formicrobiome.
a while to alter the indigenous microbiome.

Cultural Heritage
Microbiology

Culture-dependent Culture-independent

Isolation DNA / RNA

Purification Metagenomics/Metatranscriptomics
Identification

Potential Active

Purpose-driven microbial
Biochemical Processes enrichment/isolation Phylogeny
Biodeterioration Community Composition
Mechanisms Biochemical Microbial Metabolism
measurements

Figure 4. A schematic diagram showing the general framework in microbiological analyses of cultural
properties and protection.
Figure 4. A schematic diagram showing the general framework in microbiological analyses of
cultural properties
Culturing and protection.
of microorganisms is a critically-important issue in environmental microbiology and it
prevents researchers gaining further detailed insights into the biochemical function from members
Culturing
of a complex of microorganisms
microbial communityisthrough
a critically-important
isolation and pureissueculture
in environmental
techniques [73].microbiology and
On the other
it prevents researchers gaining further detailed insights into the biochemical
hand, the development and application of DNA-based molecular methods in conservation sciences function from members
of a complex
overcomes themicrobial communitylimitations
culture-dependent through isolation
to revealand
thepure culturediversity
microbial techniques and[73]. On the other
composition of
hand,
the the microbiome
whole developmentwith and greater
application
depth ofonDNA-based molecular
cultural heritage methods
of any material in conservation
type and at any sciences
time.
overcomes
Earlier the culture-dependent
molecular techniques used inlimitations
conservation to sciences
reveal the microbial
include diversity
the genetic and composition
fingerprinting analyses of
the whole
such microbiome withgradient
as DGGE/temperature greater gel
depth on cultural heritage
electrophoresis (TGGE),of any material
amplified type and
ribosomal DNAatrestriction
any time.
Earlier molecular techniques used in conservation sciences include the
analysis (ARDRA), single-strand conformation polymorphism (SSCP), terminal restriction fragment genetic fingerprinting
analyses
length such as DGGE/temperature
polymorphism gradient gelribosomal
(t-RFLR), and automated electrophoresis (TGGE),
intergenic spacer amplified
analysisribosomal
(ARISA) DNA after
restriction analysis (ARDRA), single-strand conformation polymorphism
PCR [33]. For instance, a previous study investigated the abundance and diversity of microbial (SSCP), terminal
restriction fragment
communities thrivinglength polymorphism
on damaged (t-RFLR),inand
mural paintings automated
Portugal, ribosomalculture-dependent
by combining intergenic spacer
analysis (ARISA) after PCR [33]. For instance, a previous study
methods and culture-independent methods, such as DGGE. However, it was found thatinvestigated the abundance and
the coupling
diversity
of of microbialmethods
culture-dependent communities thrivingstill
and DGGE on did
damaged mural
not yield paintings
enough in Portugal,
information by combining
to understand the
abundance and the diversity of microorganisms present on the wall paintings [76]. Because it
culture-dependent methods and culture-independent methods, such as DGGE. However, of was
the
found that the limitation
methodological coupling of andculture-dependent
sensitivity of these methods
genetic and DGGE stillanalyses,
fingerprinting did not theyyieldfailed
enoughto
information to understand the abundance and the diversity of microorganisms present
identify different members of a particular microbial group taxonomically to a satisfactory resolution on the wall
paintings
with [76]. Because of the methodological limitation and sensitivity of these genetic
confidence.
fingerprinting analyses, they failed to identify different members of a particular microbial group
taxonomically to a satisfactory resolution with confidence.
Appl. Sci. 2020, 10, 8099 7 of 15

3.1. High Through-Put Sequencing

The culture-independent methods based on PCR amplification of specific target genes, commonly
the 16S rRNA gene, which serves as an evolutionary chronometer and allows phylogenetic analysis
of obtained sequences from the microbial community [77], are becoming a routine technique used
with a sharp increase of its acceptance today. Initially, the DNA-based techniques were applied
to single colonies or libraries of PCR-amplified nucleotide sequences from environmental samples,
after successful cloning of the single PCR products, but in recent years, the application of next generation
sequencing (NGS) technology to microbial communities has revolutionized research on ecological
microbiology, enabling, at an affordable cost, both deep information and also large-scale sequencing of
DNA samples without culturing and cloning. This simplifies the analytical procedures involved and
also makes this technology accessible based on paid-for-service. Metagenomics for the characterization
of microbial community phylogeny and diversity is a new line of molecular microbiological methods
based on genomic DNA extraction directly from environmental samples and then NGS technology to
obtain the short reads of nucleotides for further assembly and analysis [78–80].
Metagenomic technologies can be either target-specific or non-specific. For the target-specific ones,
the microbial community of bacteria and archaea is revealed based on the 16S rRNA gene, and that of
eukaryotes based on the 18S rRNA or additional genes. Zhang et al. [31] investigated the bio-erosion of
the sandstone of the Royal Palace of Angkor Thom and the temple Beng Mealea using the NGS targeting
the 16S rRNA gene with samples of biofilm and exfoliated materials to obtain phylogeny and diversity
information of the bacterial community in them. The results of this study using the adhesive sampling
technique revealed that a higher bacterial diversity was detected in exfoliated sandstone materials than
in the biofilms, but the diversity of bacteria was lowest in the lower layers of the biofilm after repetitively
sampling at a selective location (discussed above). Firmicutes and Gemmatimonadetes were the
dominant phyla and were detected only in exfoliated materials, while Cyanobacteria, Chloroflexi,
and unassigned bacteria were more abundant in the biofilms. The results of this study suggested a
microbial specificity on the sandstone monuments by the nature of the sample types and the different
degree of deterioration. Biofilms grown on the surface of sandstone are in constant contact with the
underlying inorganic substratum, the shortage of organic nutrients resulting in lower diversity of the
community of heterotrophic bacteria adhering on the sandstone surface. Moreover, Cyanobacteria and
Chloriflexi were identified as the key photoautotrophic microorganisms colonizing the surface of
the sandstone. They appeared widely in the biofilms of Angkor monuments with a strong ability
for utilizing direct or indirect sunlight, resistance to the high temperature, and the ability to survive
the hypersaline environment of sandstone niche [33,38,56,62,81]. The active metabolism of these
autotrophic microorganisms results in the fixation of the inorganic CO2 as a carbon source and the
ability to modify the surface layer for retention of water, providing opportunities for subsequent
colonization by the heterotrophic bacteria and fungi [38,40,56,58,59,82]. Then, a diverse and stable
microbial community can be formed on the exfoliation sandstone materials, and the microbial activity
and abundant metabolites further accelerate the attack of the sandstone. Although the target-specific
methods can amplify and sequence marker regions of the ribosomal DNA to obtain a genus-level
picture of the microorganisms present in different environmental samples, the targeted metagenomic
analysis alone could not yield enough information about the genes and the biochemical functions in a
microbial community because of the lower depth of sequencing [83]. However, the target non-specific
metagenomics approaches, with the rapid technological advancement in high-throughput NGS, as well
as bioinformatics data processing and analysis, can extract strain-level information of the genes and the
biochemical functions in a microbial community for a better understanding of the attacking mechanisms
of microorganisms on historical materials. Microorganisms interact with underlying materials and
with other microorganisms, e.g., biofilms, and such information can be very specific in evaluating the
outcomes of the maintenance and conservation treatments on the function and community shifts of the
microbiome on cultural heritage. A recent microbiological research studied microbial phylogenetic
composition as well as biochemical capabilities of the microbiome inhabiting the sandstone surfaces
Appl. Sci. 2020, 10, 8099 8 of 15

of the Preah Vihear temple by using this non-targeted metagenomic approach. The results of this
deep metagenomics analysis provided a detailed description of the microflora present on the surface
of this cultural heritage [84]. The results indicated the presence of biochemical processes for carbon
sequestration, and nitrogen and sulfur metabolism were identified as potentially the active microbial
biochemical processes on the sandstone surface at this site. Specifically, the identified acid-producing
biochemical processes of sulfur-oxidizing bacteria and ammonia-oxidizing bacteria and archaea suggest
that the microbial flora on the sandstone surface participate in the deterioration of sandstone cultural
heritage by producing acids [32,84,85]. High-throughput NGS is a powerful method and has the
advantage of characterizing the microbial community phylogeny and diversity quickly, accurately,
and economically with a deeper data recovery [31,86]. For investigations of microbiome of cultural
heritage and the surrounding environment, where the biomass is relatively lower and the sample size
is a serious concern due to the value of the object concerned, NGS is a good candidate method for such
microbiological analysis to obtain the basic necessary data with high confidence as a first step.

3.2. Genomic DNA vs. RNA

The total microbial biomass is a useful indicator of microbial population and contamination on
solid surfaces, but the viable microbes are important estimators of microbial activity and metabolic
potential. The expression analysis of genes can yield detailed information about the metabolically-
active members and microbial metabolic state of the microbiome and about the microbe-mediated
biodeterioration process and potential, for example, following the microbial metabolic activities of
ammonia oxidation by bacteria and archaea [32,85] and sulfur oxidation [60,61] on cultural relics with
RNA. Assessment of the metabolically-active members or groups in the microbial community is a
more selective and specific approach to identifying the specific culprits than the overall information
recovered from the genomic DNA of samples.
Currently, it is necessary to determine the level of metabolic or physiological activity of specific
functional microflora in biofilm or microbiome on cultural heritage because this information is the
basis for conservators and scientists to gain a deeper understanding of the biodeterioration processes,
to monitor the effectiveness and success of the conservation treatments, and to develop alternative
and non-toxic conservation strategies to inhibit or slow down microbe-mediated biodeterioration
(Figure 4). Although real-time quantitative PCR (RT qPCR) is currently a routine tool for solving many
scientific problems, it is not useful for routine monitoring of biodeterioration and has been used only
occasionally on samples of cultural heritage. In a recent study, the community of ammonia-oxidizing
microorganisms as a biodeteriogen on the Angkor monuments, the ammonia-oxidizing archaea (AOA),
were determined to be the more active members over ammonia-oxidizing bacteria (AOB) in the
nitrifying reaction of the microbiome on Angkor monuments. They can play an important role in
biodeterioration according to the results from the RT qPCR analyzing the expression of amoA gene of
samples [32]. Further confirmation of the biochemical capabilities are still expected to establish the
microbial transformation processes responsible for destruction of materials (see next section).
Gene-by-gene molecular analysis is too time-consuming and requires a high level of technical skill
in conservation science. As a result, it is not cost-effective in application. Recent metatranscriptomics
approaches that rely on NGS platforms offer more possibilities for understanding the metabolic
pathways and the activity of the specific metabolic processes within microbial communities [30,83,87].
However, metatranscriptomics approaches applied to the study of biodeterioration of cultural heritage
are still in their infancy and their application in conservation science is not yet widely available.
In general, the costs for molecular analysis are going down rapidly and operational issues with RNA
protection and extraction are also improving. Bioinformatics tools to process the metatranscriptome
datasets are also becoming routine for processing to microbial communities and gene assembly
associated with biodeterioration.
Appl. Sci. 2020, 10, 8099 9 of 15

4. Biochemical Functional Approaches

Both growth on surface as biofilms and specific metabolic activity (i.e., acidification) of the active
microorganisms are important information for the understanding and prediction of damage to the
materials of cultural heritage. Earlier investigations suffered from the isolation of limited numbers
of cultured bacteria in pure cultures from the general community of microorganisms due to the
technical means available. There were several early attempts to quantify the activity of the microbiome
based on chemical reactions [30,88]. Detection and quantifying ATP can be used as an estimation
of biological activity on surfaces like paper, paintings, or other materials in swab samples using the
commercially-available products of luminometers [89,90]. However, the active microbial groups and
members on cultural heritage may not be the directly destructive ones responsible for the damage
caused to the cultural heritage and the general purpose-driven biochemical analysis of the samples from
cultural heritage only provides limited insights because a close relationship between the destruction
and specific microbial group cannot be established.
The “tangible” or physical cultural heritage, no matter whether moveable artifacts or
immovable historic sites, is in a constant state of chemical transformation and equilibrium with
the surrounding environmental conditions [91] and selective biochemical processes accelerate the
chemical transformation of the underlying materials given the colonization by microorganisms.
Selective microbial groups carry out the important biochemical transformation reactions with high
specificity regulated by relevant genes and the corresponding enzymes/proteins. It is known that
autotrophic microorganisms are pioneering organisms that colonized the bare surfaces of sandstone
when the cultural heritage was initially built, but the roles of these microorganisms are not
straight-forwardly detrimental because their protective role for the sandstone, for example from lichen,
was significant in the beginning [40]. After sequestration of atmospheric CO2 onto the surface as biomass,
the physical surface properties of the heritage are significantly altered, allowing for diversification of the
microbial community with different physiological functions and also for accumulation of pollutants to
promote the degradative microorganisms [63]. Information on these has been reviewed recently [40,92].
More recently, NO3 − production and accumulation of the nitrogen cycle have been widely observed
and confirmed on Angkor sandstone monuments under the tropical climate in Cambodia [32,85].
The concentration of NO3 − is positively correlated with the acidity and pH value, among the different
environmental variables of these sandstone monuments. The constant acidification on the cultural
heritage initiates and accelerates the dissolution of mineral constituents in the sandstone and produces
the phenomenon of salt attack by crystallization/efflorescence on/in the substratum material which leads
to structural weakening and crack formation of the cultural heritage [16,29,30,35,70,93]. After successive
sampling over a 3-year period and analyses of the ammonia-oxidizing archaea (AOA) and bacteria
(AOB) by both genomic DNA and RNA from samples for real-time PCR, AOA was apparently
higher than AOB on these sandstone monuments and detection of NO3 − was successful [32,85].
Furthermore, a stable isotope tracer experiment by addition of the 15 N in the chemical forms of NH4 +
or NO3 − was initiated and incubated with samples collected from several Angkor monuments to
quantify the nitrogen transformation rate from nitrate removal as nitrogen gas and determine the
contribution of the microbial community to the removal of available nitrate. The results of this study
revealed that the heterotrophic denitrification (NO3 − →NO2 − →NO→N2 ) activity in the microbiome of
the sandstone monument was low, and the activity of anammox (NH4 + +NO2 − →N2 ) in the sample
from the monument was negligible [84]. According to data in this study, the nitrate removal reaction
of denitrification was active, but not strong enough to remove the produced and accumulated NO3 −
because ammonia oxidation is aerobic while denitrification is anoxic. The physical conditions of
available oxygen most likely play the regulation role, resulting in the accumulation of nitrate on the
Angkorian sandstone.
Sulfur is also involved in the sandstone attack through microbial transformation of S at different
valent states. Fusarium sp. and Mycobacterium spp. were isolated from decayed materials of the
sandstone monuments and elucidated their metabolic capability of sulfur oxidation to acidic sulphate
Appl. Sci. 2020, 10, 8099 10 of 15

via chemolithoautotrophic metabolism in laboratory experiments [61,62]. The metabolic products of

these sulfur-oxidizing bacteria are capable of producing inorganic acid through oxidation of S and the
acidity in the form of H+ interacts with minerals causing dissolution and re-crystallization regulated
through water retention and loss by natural force, resulting in structural weakening of the stone
material integrity and disintegration [29,40,70]. These findings suggested that these sulfur-oxidizing
Fusarium sp. and Mycobacterium spp. participate in the deterioration process (i.e., acidification) of
the sandstone monuments under tropical conditions. The accumulation of acids, NO3 − and SO4 2− ,
which are corrosive to the inorganic material substratum of the cultural heritage, can be observed by
microbially-mediated biochemical transformations.
As the former section stated, NGS techniques can provide thorough information of the
microbial community and composition inhabiting cultural heritage to allow for identifying of the
microorganisms without culturing them, which cannot be achieved by a culture-dependent approach.
However, the involvement of the new microorganisms in the biodeterioration needs further careful
verification even if high percentages of sequence reads with a metabolic potential are detected.
The results of NGS-based investigations can provide useful information for the purpose-driven
biochemical functional analysis and identify the physiological characteristics of the abundant genes
and associated groups for developing culturing strategies to enrich and isolate the implicated members
for further verification of their biochemical and ecophysiological roles in deterioration (Figure 4).
Therefore, NGS technology can be employed to obtain basic information to assist in further research
directions and strategies to directly confirm the scientific questions prior to culture-based strategies or
activity-based biochemical functional analysis. It is also important to use NGS to gain an overview
or summary of the whole microbial community prior to a genuine scientific question being executed.
In doing this way, culture-dependent and -independent methods can be integrated effectively to
advance scientific research on cultural heritage and also provide useful understanding for protection
and management.

5. Summary and Future Perspectives

Current research addressing the issue of biodeterioration of cultural heritage needs to focus
on sampling methods and appropriate analytical techniques used afterward. Application of
non-invasive/non-destructive sampling strategies is required in microbiological studies for conservation.
The NGS-based metagenomics and metatransriptomics should be applied to the study of biodeterioration
of cultural heritage for the microbial structure (especially spatial distribution structure) of the
microbiome and the potential of specific microbial groups and microbe-mediated transformation
processes contributing to biodeterioration. The NGS-based results can provide sound basis for the
purpose-driven biochemical functional analysis or develop enrichment and culturing strategies to
isolate the implicated groups of microorganisms with significant roles in biodeterioration. There are
apparent challenges for investigations to develop the purpose-specific biochemical functional analysis
for measuring the valid deterioration effects of microbiome and for enhancing knowledge of cultivation
of microorganisms from a specific material.
Delineation of the processes and mechanisms involved for (bio)deterioration should be the basis
for understanding the current status with a selective cultural heritage. When the destructive reactions
are known and also the specific microorganisms responsible for the processes, remediation strategies
and management plan can be proposed accordingly [94]. Sulfur-driving acid production can be
dealt with from restriction on fuel quality and air quality mandate in policy and management while
intervention on site can be carried out through mineralization to immobilize produced SO4 2− . NO3 − is
an issue that is more difficult to address than SO4 2− , but an effective way to deal with it should be to
focus on the source of NH4 + to eliminate its contribution from the beginning. In addition, new emerging
science on materials can also take an important role in the effective protection and management of
culture heritage to decrease the detrimental contribution from microorganisms and their biochemical
Appl. Sci. 2020, 10, 8099 11 of 15

capabilities. Overall, protection of cultural heritage is a complex subject and synergies of different
subjects will contribute to the advancement of this field for both basic knowledge and also applications.

Author Contributions: Conceptualization, W.L. and J.-D.G.; writing—original draft preparation, X.D.; writing—
review and editing, X.D., W.L. and J.-D.G.; supervision, J.-D.G.; project administration and funding acquisition,
W.L. and J.-D.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Shenzhen Scientific Project Fund (JCYJ20170410103015603), and Hong Kong
RGC GRF Grant (No. 17302119).
Acknowledgments: This project was financially supported by Shenzhen Scientific Project Fund
(JCYJ20170410103015603), and Hong Kong RGC GRF Grant (No. 17302119). We would like to thank the
Apsara Authority of the Kingdom of Cambodia for permission to conduct sampling and research for more than
a decade, Ka Hong Cheung for the SEM in Figure 1, and Lin Gao for help with the photographs presented in
Figure 2.
Conflicts of Interest: The authors declare no conflict of interest.

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